Il 10

Manufactured by Mabtech

Sourced in Sweden, United States

IL-10 is a recombinant protein that functions as an interleukin. It is a cytokine with immunoregulatory properties.

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11 protocols using il 10

HCMV-specific CD4+ T Cell Suppression

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PBMC were depleted of CD8+ T cells by MACS using anti-CD8+ direct beads (Miltenyi Biotec), according to manufacturer’s instructions and separated on an AutoMACS Pro. The resulting CD4+ T cell & Antigen Presenting Cell (APC) PBMC were resuspended in either X-VIVO 15, X-VIVO 15 with 4ng/ml TGF-β and 10ng/ml IL-10 (both Miltenyi Biotec), neat Mock infected monocyte secretome, neat UV irradiated infected monocyte secretome or neat Latent Infected Monocyte secretome. The cells were then plated in 48-well tissue culture plates and incubated overnight at 37°C in a humidified CO₂ atmosphere. After 24 hours incubation, the cells were stimulated with 1µg/ml anti-CD3 and 0.5µg/ml anti-CD28 (both Mabtech AB) and overlapping peptide pools for HCMV proteins (43 (link)) resulting in a 1:2 dilution of the secretomes and TGF-β/IL-10 mix. Following a further 24-hour incubation at 37°C in a humidified CO₂ atmosphere, the plates were centrifuged, and supernatants harvested and then analyzed for the production of IFN-γ by ELISA. Full details of proliferation assays used to measure whether latent secretomes can suppress CD4+ T cells can be found in the supplementary material (Section 1.8).

Jackson S.E., Chen K.C., Groves I.J., Sedikides G.X., Gandhi A., Houldcroft C.J., Poole E.L., Montanuy I., Mason G.M., Okecha G., Reeves M.B., Sinclair J.H, & Wills M.R. (2021). Latent Cytomegalovirus-Driven Recruitment of Activated CD4+ T Cells Promotes Virus Reactivation. Frontiers in Immunology, 12, 657945.

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T-cell Response Analysis by ELISpot

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Elispot analysis was performed after 3 days of DC/T-cell co-culture. After that CD4⁺/OT-II T-cells were counted and plated again over night at 37°C in a 96-well/ELISpot-plate (Cellular Technology Limited, CTL, ImmunoSpot, Cleveland, USA), with 6 h-LPS-treated DC-OVA, just OVA or TRP as controls, and PMA+Ionomycin. To monitor unspecific signals and background, T-cells, DC-OVA, DC-TRP, Lcn2^−/− DCs and WT DCs, were also co-cultured. We used ELISpot plates (Millipore, Massachusetts, USA) for IFN-γ, IL-10 and IL-17A (from Mabtech, Sweden), following the ImmunoSpot protocol and recommendations. Spots were counted using ImmunoSpot 5.0 software. Histograms are the results of the spots from T-cell+WT DC-OVA or T-cell+Lcn2^−/− DC-OVA calculated after subtracting the spots of the controls.

Floderer M., Prchal-Murphy M, & Vizzardelli C. (2014). Dendritic Cell-Secreted Lipocalin2 Induces CD8+ T-Cell Apoptosis, Contributes to T-Cell Priming and Leads to a TH1 Phenotype. PLoS ONE, 9(7), e101881.

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ELISPOT Assay for IFNγ and IL-10 Producing Cells

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Frequencies of IFNγ or IL-10 producing cells were determined in PBMC-sorted DR^hi or MDSC co-cultured in ELISPOT plates coated with capture antibodies for IFNγ and IL-10, respectively. Cytokine producing cells were detected using kits for IFNγ (BD Biosciences) and IL-10 (Mabtech) according to manufacturer’s instructions. Air-dried spots were counted with automated reader S5 UV Lite Analyzer (Immunospot). For some experiments, PBMC from CMV(+) HIV-infected or HIV-uninfected donors were cultured in the presence of 1 μg/ml of CMVpp65 for 48–72 hrs, CD4⁺CX3CR1⁻ and CD4⁺CX3CR1⁺ cells were sorted and cultured in ELISPOT plates coated with capture antibody for IFNγ. Cytokine producing cells were detected as above.

Garg A., Trout R, & Spector S.A. (2017). Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4. Scientific Reports, 7, 44485.

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Cytokine and Chemokine Quantification

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Cytokines and chemokines were measured in cell supernatant and plasma by ELISA (IL-6 and TNF: R&D systems, Minneapolis, MN; IL-10: Mabtech, Nacka Strand, Sweden) or by Luminex (Mouse Custom ProcartaPlex 17-plex: ENA-78/CXCL5, G-CSF, IFNγ, IL-1α, IL-1β, IL-3, IL-6, IL-10, IL-12p40, IL-17A, IL-18, IP-10/CXCL10, KC/CXCL1, MCP-1/CCL2, MIP-1α/CCL3, MIP-2/CXCL2, TNF) (Invitrogen) and recorded with a bioplex 200 (Bio-Rad, Hercules, CA) (60 (link)).

Heinonen T., Ciarlo E., Rigoni E., Regina J., Le Roy D, & Roger T. (2019). Dual Deletion of the Sirtuins SIRT2 and SIRT3 Impacts on Metabolism and Inflammatory Responses of Macrophages and Protects From Endotoxemia. Frontiers in Immunology, 10, 2713.

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Cytokine Quantification from Mouse Colon

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Colon segments of approximatively 2 cm were placed in Lysing Matrix Tubes (MP Biomedicals) containing 1.4 mm-diameter ceramic spheres and 1 mL of RIPA buffer (1% Igepa, 0.5% deoxycholic acid and 0.1% SDS in Tris buffered saline solution 1×; pH 7.4) with protease inhibitors (Roche Diagnostics). The tubes were shaken at 6 m/s two times for 20 s each in a FastPrep (MP Biomedicals). After centrifugation at 10,000×g for 4 min (4 °C), the supernatants were recovered and protein concentrations were assessed using a Coomassie protein assay kit (Thermo Scientific) according to the manufacturer’s instructions. All supernatants were normalized at 1 mg/mL of protein with PBS and stored at − 80 °C until processing. ELISAs were performed on the supernatants to quantify the following mouse cytokines according to the manufacturer’s instructions: IL-10, IL-17A, TNFα and IFNγ (Mabtech, Nacka Strand, Sweden); IL-6, IL-1β and TGFβ (R&D Systems, Minneapolis, MN, USA).

Evariste L., Lamas B., Ellero-Simatos S., Khoury L., Cartier C., Gaultier E., Chassaing B., Feltin N., Devoille L., Favre G., Audebert M, & Houdeau E. (2023). A 90-day oral exposure to food-grade gold at relevant human doses impacts the gut microbiota and the local immune system in a sex-dependent manner in mice. Particle and Fibre Toxicology, 20, 27.

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Cytokine Profiling of Murine Immune Cells

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MLNs and spleens were sieved through a 70-μm cell strainer (BD Difco) in complete RPMI 1640 medium (10% heat-inactivated FCS, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin; Sigma-Aldrich), and 1 × 10⁶ cells per well were cultured (37 °C, 10% CO₂) for 48 h with stimulation by phorbol 12-myristate 13-acetate (PMA, 50 ng/ml; Sigma-Aldrich) and ionomycin (1 μM; Sigma-Aldrich). The culture supernatant was frozen at –80 °C until processing. To measure cytokine levels in the colonic explants, tissues from the medial colon were isolated and rinsed in phosphate-buffered saline (PBS; Gibco, Paisley, UK). The colonic explants were cultured (37 °C, 10% CO₂) overnight in 24-well tissue culture plates (Costar, Corning, Amsterdam, the Netherlands) in l ml of complete RPMI 1640 medium. The culture supernatants were collected and stored at –80 °C until processing. ELISAs were performed on the supernatants to quantify the following mouse cytokines according to the manufacturer’s instructions: IL-10, IL-17A, and IFN-γ (Mabtech, Nacka Strand, Sweden); IL-22 (eBioscience, San Diego, CA, USA); and IL-6 (R&D Systems, Minneapolis, MN, USA). For the colonic explants, cytokine concentrations were normalized according to the dry weight of each colonic explant.

Lamas B., Richard M.L., Leducq V., Pham H.P., Michel M.L., Da Costa G., Bridonneau C., Jegou S., Hoffmann T.W., Natividad J.M., Brot L., Taleb S., Couturier-Maillard A., Nion-Larmurier I., Merabtene F., Seksik P., Bourrier A., Cosnes J., Ryffel B., Beaugerie L., Launay J.M., Langella P., Xavier R.J, & Sokol H. (2016). CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands. Nature medicine, 22(6), 598-605.

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Cytokine Profile of Phage Preparations

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ELISA was performed on purified phage preparations of different concentrations (10⁹, 10⁷, and 10⁵ PFU/well) using commercially available kits IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α (Mabtech AB, Stockholm, Sweden) according to the manufacturer’s instructions. Optical density (OD) was determined at 405 nm on a micro-plate reader (Molecular Devices Corp, Sunnyvale, CA, USA). Data were analyzed in the SoftMax Pro 5.2 rev C software package (Molecular Devices Corp, Sunnyvale, CA, USA).

Khan Mirzaei M., Haileselassie Y., Navis M., Cooper C., Sverremark-Ekström E, & Nilsson A.S. (2016). Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro. Frontiers in Microbiology, 7, 437.

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Quantitative Cytokine and PGE2 Analysis

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Quantitative analysis of PGE2 (Enzo life science, USA), IL-10, IFN-γ and IL-17 (Mabtech, Sweden) was performed by commercially available ELISA kits. To determine the amount of PGE2 and IL-10 produced by MSCs and MSCs-NPs, 5×10⁵ cells/well were cultured in 2 ml DMEM or 2 ml neurobasal media, respectively. After 24 hours and 48 hours supernatants were collected. The concentrations of cytokines and PGE2 were reported after reduction from basal levels. Serums and spleen cells were also isolated from EAE mice transplanted with MSCs, MSCs-NPs, combination of MSCs+MSCs-NPs and control mice with EAE on day 45. Isolated splenocytes were cultured (2×10⁵ cells/well) in triplicate in medium supplemented with 20 μg/ml MOG_35–55 and the supernatants were collected after 72 hours.

Nasri F., Mohtasebi M.S., Hashemi E., Zarrabi M., Gholijani N, & Sarvestani E.K. (2018). Therapeutic Efficacy of Mesenchymal Stem Cells and Mesenchymal Stem Cells-derived Neural Progenitors in Experimental Autoimmune Encephalomyelitis. International Journal of Stem Cells, 11(1), 68-77.

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Cytokine and Antibody Quantification

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The cytokines IL-5, IL-10, IL-12p70, IL-17a or IFNγ (Mabtech, Nacka Strand, Sweden) and TSLP (eBiosciences, Paris, France) were quantified by enzyme-linked immunosorbent assay according to the manufacturer’s instructions. Individual mouse sera were assayed for IgE and IgG1 (eBiosciences) according to the manufacturer’s instructions.

Remot A., Descamps D., Noordine M.L., Boukadiri A., Mathieu E., Robert V., Riffault S., Lambrecht B., Langella P., Hammad H, & Thomas M. (2017). Bacteria isolated from lung modulate asthma susceptibility in mice. The ISME Journal, 11(5), 1061-1074.

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Cytokine Secretion Measurement Protocol

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Secreted levels of the cytokines IL-1ra (R&D Systems-BioTechne), IL-1β, IL-6, IL-10, IL-17A and IFN-γ (MabTech AB) were measured in cell culture supernatants using sandwich ELISA kits according to the manufacturer’s instructions. Absorbance was measured at a wavelength of 405 nm using a micro-plate reader (Molecular Devices Corp.) and results analysed using SoftMax Pro 5.2 rev C (Molecular Devices Corp.).

Mata Forsberg M., Björkander S., Pang Y., Lundqvist L., Ndi M., Ott M., Escribá I.B., Jaeger M.C., Roos S, & Sverremark-Ekström E. (2019). Extracellular Membrane Vesicles from Lactobacilli Dampen IFN-γ Responses in a Monocyte-Dependent Manner. Scientific Reports, 9, 17109.

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